Closed system device and methods for gas permeable cell culture process

ABSTRACT

Novel methods and apparatus are disclosed for cell culture and cell recovery. The methods and apparatus simplify the process of cell separation from media, minimize potential damage to gas permeable devices during fluid handling, and allow closed system automated cell culture and cell recovery from gas permeable devices.

RELATED APPLICATIONS

This application is a Continuation of U.S. Pat. No. 11,155,778, filedOct. 5, 2017, which is a Continuation of U.S. patent application Ser.No. 14/313,702, filed Jun. 24, 2014, now U.S. Pat. No. 9,840,692, whichclaims the benefit of U.S. Provisional Patent Application No.61/838,730, entitled “CLOSED SYSTEM DEVICE AND METHODS FOR GAS PERMEABLECELL CULTURE PROCESS”, and filed Jun. 24, 2013, said application beinghereby fully incorporated herein by reference. Additionally, co-pendingU.S. patent application Ser. No. 10/961,814 (hereinafter “814”), U.S.patent application Ser. No. 11/952,848 (hereinafter “848”), U.S. patentapplication Ser. No. 12/963,597 (hereinafter “597”), U.S. patentapplication Ser. No. 13/475,700 (hereinafter “700”), and U.S. patentapplication Ser. No. 13/493,768 (hereinafter “768”) are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The technical field of the invention relates to static cell culturemethods and devices that efficiently expand cells within novel gaspermeable cell culture devices and allow fluids to be added and removedfrom the culture system with little or no contamination risk, little orno loss of cells, and little or no distortion of gas permeable surfaces.

DISCUSSION OF LIMITATIONS OF CONVENTIONAL TECHNOLOGIES DESCRIBED INRELATED ART

T cell therapy, adoptive immunotherapy, and adoptive cell therapy referto powerful methods for treating various diseases that include expandingcells of the immune system in vitro and subsequently infusing theexpanded cells into a patient to fight disease. For these forms oftherapy to reach a wide segment of society, the process of expanding thea population of cells and collecting the cells needs to be costeffective, practical, not prone to cell loss, and have minimal to nocontamination risk.

To date, there is not a cost effective and practical system forpreparing and storing the cells produced in T cell therapy, adoptiveimmunotherapy, and adoptive cell therapy applications that is notsubject to contamination and also does not require a significant amountof effort to separate cells from media after they have been produced. Akey element of making the T cell production process practical for FDAapproval is to minimize and even eliminate the chance for contamination,while minimizing process complexity. In the normal lexicon in thisfield, a culture process that is generally closed to contamination iscommonly referred to as a “closed system.” WAVE Bioreactor™, OriGenPermaLife™ bags, and/or VueLife® bags are the devices adapted for closedsystem T cell production. Bags have flexible housings that are able tocollapse as media and cells are removed, typically by squeezing the bag,relying on gravity, and or by withdrawing media by peristaltic pumps.WAVE Bioreactor™ relies on peristaltic pumps to remove media and cells.

Recently G-Rex™ cell culture devices have become popular for T cellculture based on numerous advantages relative to WAVE Bioreactor™,OriGen PermaLife™ bags, and VueLife® bags. One of the advantages isdescribed in co-pending '570 is the ability to remove the vast majorityof media prior to collection of cells to minimize the amount of time andeffort needed to separate cells from media. However, it has since beendiscovered that state-of-the-art methods of media and cell removal whichrely the aforementioned methods do not work properly with G-Rex™ culturedevices. G-Rex devices do not collapse as media is removed as do bagsand use of peristaltic pumps to remove media from G-Rex devices puts theplanar gas permeable surface in a state where it moves from itshorizontal culture position and is pulled into the internal volume ofthe device by a vacuum that forms in the device. Attempts to prevent avacuum from forming by use of larger and larger surface area ventfilters have not led to a feasible configuration that allows the gaspermeable surface to remain in a planar and/or horizontal position.Having the membrane pulled out of its planar and/or horizontal state isdetrimental to the production process and creates the potential forcells to be lost when media volume is diminished or for leaks to formwhich can lead to contamination of the culture and/or expose workers tobiohazards.

Thus, in order to simplify the process of cell recovery in a closedsystem manner, particularly for the field of T cell therapy, thereexists a need to create a novel method of closed system fluid handlingfor gas permeable cell culture devices, including those disclosed in'814, '848, '597, '700, and '768.

SUMMARY OF THE INVENTION

Certain embodiments are disclosed that allow gas to displace a portionof an initial volume of media into a waste receptacle prior todisplacing cells and a residual volume of media into a cell collectionvessel.

One such embodiment discloses an apparatus for removing media from acell culture device comprising a gas delivery component that is capableof connecting to a filter that is connected to a gas permeable cellculture device, said gas delivery component capable of delivering gasinto said gas permeable cell culture device by way of said filter, afirst fluid detection component that is capable of determining when thefluid moving within a media removal conduit that is connected to saidgas permeable cell culture device changes from liquid to gas, said firstfluid detection component capable of sending a signal to a first fluidflow control component that is capable of terminating fluid flow throughsaid media removal conduit.

One such embodiment discloses an apparatus including a second fluiddetection component that is capable of determining when fluid movingwithin a cell removal conduit that is connected to said gas permeablecell culture device changes from liquid to gas, said second fluiddetection component capable of sending a signal to a second fluid flowcontrol component capable of terminating fluid flow through said cellremoval conduit.

One such embodiment discloses use of an apparatus for reducing thevolume of liquid media in a gas permeable cell culture device thatcontains cells and media in order to increase the concentration of cellsper milliliter of media by connecting a gas delivery component to afilter that is connected to the gas permeable cell culture device, acell culture device including a media removal conduit, connecting afirst fluid detection component to the media removal conduit, connectinga first fluid flow control component to the media removal conduit,initiating gas delivery from the gas delivery component, whereby gasmoves into the cell culture device, the gas displaces media from thecell culture device into a media collection vessel connected to themedia removal conduit, the first fluid detection component determineswhen the fluid that is moving through the media removal conduit haschanged from liquid to gas and upon making that determination the firstfluid detection component sends a signal to the first fluid flow controlcomponent, and upon the first fluid flow control component receiving thesignal the first flow control component terminates the flow of fluidthrough the media removal conduit.

One such embodiment discloses use of an apparatus for collecting cellswherein after a first flow control component has terminated the flow offluid through a media removal conduit, a cell collection vessel isconnected to a media removal conduit, the media removal conduit having amedia removal opening in contact with media, first flow controlcomponent opens the flow of fluid through the media removal conduit, gasdelivered from the gas delivery component moves into the cell culturedevice, media and cells move through the media removal conduit into thecell collection vessel, the first fluid detection component determineswhen the fluid that is moving through the media removal conduit haschanged from liquid to gas and sends a signal to the first fluid flowcontrol component, and upon receiving the signal the first flow controlcomponent terminates the flow of fluid through the media removalconduit.

One such embodiment discloses increasing the number of cells permilliliter of media in a gas permeable cell culture device by connectinga gas delivery component to a filter that is connected to a gaspermeable cell culture device containing liquid media and cells, atleast a portion of the cells in contact with a growth surface within thecell culture device, the cell culture device including a media removalconduit and a cell removal conduit, connecting the first fluid detectioncomponent to the media removal conduit, connecting the first fluid flowcontrol component to the media removal conduit, initiating gas deliveryfrom the gas delivery component whereby gas moves into the cell culturedevice and the gas displaces media from the cell culture device into amedia collection vessel connected to the media removal conduit, thefirst fluid detection component determining when the fluid that ismoving through the media removal conduit has changed from liquid to gasand upon making that determination, the first fluid detection componentsends a signal to the first fluid flow control component, and uponreceiving said signal the first flow control component terminates theflow of fluid through the media removal conduit, and removing cells fromthe gas permeable cell culture device by connecting the second fluiddetection component to the cell removal conduit, connecting the secondfluid flow control component to the cell removal conduit, initiating gasdelivery from the gas delivery component, whereby gas moves into thecell culture device and displaces media and cells by way of the cellremoval conduit from the cell culture device into a cell collectionvessel connected to the cell removal conduit, the second fluid detectioncomponent determining when the fluid that is moving through the cellremoval conduit has changed from liquid to gas and when that isdetermined the second fluid detection component sends a signal to thesecond fluid flow control component and upon receiving said signal, thesecond flow control component terminates the flow of fluid through saidcell removal conduit.

One such embodiment discloses collecting concentrated cells from a gaspermeable cell culture device wherein after a second flow controlcomponent has terminated the flow of fluid through a cell removalconduit, the cell culture device is vented to atmosphere, first flowcontrol component is opened to allow the flow of fluid through the mediaremoval conduit, media is moved through the media removal conduit intothe cell culture device, the first flow control component is closed, themedia is agitated to move cells into the media, and the cell culturedevice is no longer vented to atmosphere and gas is delivered from thegas delivery component into the cell culture device and displaces mediaand cells from the cell culture device into a cell collection vesselconnected to the cell removal conduit, the second fluid detectioncomponent determining when the fluid that is moving through the cellremoval conduit has changed from liquid to gas and upon making thatdetermination the second fluid detection component sends a signal to thesecond fluid flow control component and upon receiving the signal, thesecond flow control component terminates the flow of fluid through saidcell removal conduit.

One such embodiment discloses increasing the number of cells permilliliter of media in a gas permeable cell culture device that containsa first volume of media and cells residing on a growth surface byreducing the volume of media in order to increase the concentration ofcells per milliliter of media by connecting a gas delivery component toa filter that is connected to the cell culture device, the cell culturedevice including a media removal conduit, connecting a first fluiddetection component to the media removal conduit, connecting a firstfluid flow control component to a media removal conduit, initiating gasdelivery from the gas delivery component, whereby gas moves into thecell culture device, the gas displaces media from the cell culturedevice into a media collection vessel connected to the media removalconduit, the first fluid detection component determines when the fluidthat is moving through the media removal conduit has changed from mediato gas and upon making that determination the first fluid detectioncomponent sends a signal to the first fluid flow control component, andupon said first fluid flow control component receiving the signal thefirst flow control component terminates the flow of fluid through themedia removal conduit leaving a residual volume of media and cellswithin said cell culture device, the residual volume of media being lessthan the first volume of media.

One such embodiment discloses concentrating cells within a gas permeablecell culture device wherein after a first flow control component hasterminated the flow of fluid through a media removal conduit, a cellcollection vessel is connected to the media removal conduit, and withmedia in contact with a media removal opening of the media removalconduit first flow control component is opened to allow the flow offluid through the media removal conduit, gas delivered from the gasdelivery component moves into the cell culture device displacing theresidual media volume and cells through the media removal conduit andinto the cell collection vessel, the first fluid detection componentdetermines when the fluid that is moving through the media removalconduit has changed from media to gas and sends a signal to the firstfluid flow control component, and upon receiving the signal the firstflow control component terminates the flow of fluid through said mediaremoval conduit.

One such embodiment discloses collecting cells from a gas permeable cellculture device comprising a first step of reducing a first volume ofmedia within a gas permeable cell culture device that contains cells inorder to increase the concentration of cells per milliliter byconnecting a gas delivery component to a filter that is connected to agas permeable cell culture device containing media and cells, the cellculture device including a media removal conduit and a cell removalconduit, connecting a first fluid detection component to the mediaremoval conduit, connecting a first fluid flow control component to themedia removal conduit, and with at least a portion of the cells residingon a growth surface initiating gas delivery from the gas deliverycomponent whereby gas moves into the cell culture device and the gasdisplaces media from the cell culture device into a media collectionvessel connected to the media removal conduit, the first fluid detectioncomponent determining when the fluid that is moving through the mediaremoval conduit has changed from media to gas and upon making thatdetermination the first fluid detection component sends a signal to afirst fluid flow control component, and upon receiving the signal thefirst flow control component terminates the flow of fluid through themedia removal conduit leaving a residual volume of media and cellswithin the cell culture device, the residual volume of media being lessthan said first volume of media, and a second step of removing cellsfrom the gas permeable cell culture device by connecting a second fluiddetection component to the cell removal conduit, connecting a secondfluid flow control component to the cell removal conduit, and with cellsdistributed throughout said residual volume of media initiating gasdelivery from a gas delivery component, whereby gas moves into the cellculture device and displaces media and cells from the cell culturedevice into a cell collection vessel connected to said cell removalconduit, the second fluid detection component determining when the fluidthat is moving through the cell removal conduit has changed from mediato gas and when that determination is made the second fluid detectioncomponent sends a signal to the second fluid flow control component andupon receiving the signal, the second flow control component terminatesthe flow of fluid through the cell removal conduit.

One such embodiment discloses collecting cells including an additionalstep of rinsing the cell culture device to gather additional cells thatmay have been left in the cell culture device and/or the cell removalconduit after the second flow control component has terminated the flowof fluid through the cell removal conduit further comprising initiatingthe flow of liquid through the media removal conduit when the cellculture device is vented to atmosphere, the liquid moving through saidmedia removal conduit into the cell culture device, agitating the liquidto move cells within the cell culture device into the liquid, andinitiating gas delivery from the gas delivery component into the cellculture device, the gas displacing liquid and cells from the cellculture device into the cell collection vessel via the cell removalconduit, the second fluid detection component determining when the fluidthat is moving through the cell removal conduit has changed from liquidto gas and when that determination is made the second fluid detectioncomponent sends a signal to the second fluid flow control component andupon receiving the signal, the second flow control component terminatesthe flow of fluid through the cell removal conduit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a cross-sectional view of a gas permeable cell culturedevice.

FIG. 1B shows a cross-sectional view of a gas permeable cell culturedevice with a waste receptacle attached to it.

FIG. 1C shows a cross-sectional view of a gas permeable cell culturedevice with a waste receptacle attached to it, a peristaltic pump movingmedia into the waste receptacle, and a growth surface moving out of ahorizontal position.

FIG. 1D shows a cross-sectional view of a gas permeable cell culturedevice with a waste receptacle attached to it, a peristaltic pump movingmedia into the waste receptacle, a growth surface moving out of ahorizontal position, and cells entering the waste receptacle.

FIG. 2A shows a cross-sectional view of a gas permeable cell culturedevice with a waste receptacle attached to it.

FIG. 2B shows a cross-sectional view of a gas permeable cell culturedevice with a waste receptacle attached to it, a peristaltic pump movingmedia into the waste receptacle, a growth surface remaining in ahorizontal position, and an initial media volume having been reduced toa residual media volume in which cells reside.

FIG. 2C shows a cross-sectional view of a gas permeable cell culturedevice after being oriented to a cell recovery position such that cellsand residual media can be removed.

FIG. 2D shows a cross-sectional view of a gas permeable cell culturedevice after being oriented to a cell recovery position and after cellsand residual media have been moved to a cell collection receptacle.

FIG. 3 shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit opening.

FIG. 4A shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit openingpositioned in a media removal location.

FIG. 4B shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit openingpositioned in a cell removal location.

FIG. 5A shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit openingpositioned in a cell removal location.

FIG. 5B shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit openingpositioned in a cell removal location.

FIG. 5C shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a media removal conduit openingpositioned in a cell removal location and where the cell removal conduitopening resides in a pocket of the growth surface.

FIG. 6A shows a cross-sectional view of a gas permeable cell culturedevice with a media removal conduit and a cell removal conduit.

FIG. 6B shows a cross-sectional view of a gas permeable cell culturedevice after gas has been pushed into the device and media has beenremoved via the media removal conduit.

FIG. 6C shows a cross-sectional view of a gas permeable cell culturedevice after gas has been pushed into the device and media has beenremoved via the media removal conduit and moved into a waste collectionreceptacle.

FIG. 6D shows a cross-sectional view of a gas permeable cell culturedevice after gas has been pushed into the device and cells and aresidual volume of media have be removed via a cell removal conduit andmoved into a cell collection receptacle.

FIG. 7 shows a schematic view of a gas permeable cell culture deviceinterfacing with equipment to automate the removal of media and cells.

FIG. 8 shows a cross-sectional view of a growth surface interlocked intoa growth surface support.

FIG. 9 shows a cross-sectional view of a growth surface molded onto agrowth surface support.

FIG. 10 shows a cross-sectional view of a gas permeable cell culturedevice and a process by which the growth surface remains in a planarposition during media and cell removal.

FIG. 11 shows a cross-sectional view of a gas permeable cell culturedevice and a process by which the growth surface remains in a planarposition during media and cell removal.

FIG. 12A and FIG. 12B show a cross-sectional view of a gas permeablecell culture device and a process by which the growth surface remains ina planar position during media and cell removal.

FIG. 13A, FIG. 13B, and FIG. 13C show a cross-sectional view of a gaspermeable cell culture device and a process by which the growth surfaceremains in a planar position during media and cell removal.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, unless otherwise specified, the followinggeneral considerations apply. Preferably when using the devices andmethods disclosed herein, cells reside upon a gas permeable surface in auniformly distributed state. Skilled artisans are advised to select gaspermeable material that comports to that typically used in the cellculture field. Preferably, the gas permeable material is liquidimpermeable. Additional guidance for the types of gas permeable surfacesthat can be used is also found in co-pending '814, '848, '597, '700, and'768. When any type of non-adherent animal cells are the intended cellsto be cultured, the gas permeable surface is preferably non-porous,liquid impermeable, and hydrophobic. Most preferably it is comprised ofsilicone and has a thickness between 0.001 inches and 0.024 inches. Inthe case of T cells in particular, silicone is a preferred material.Furthermore, the gas permeable material preferably resides in ahorizontal position during culture in order for cells to gravitate tothe gas permeable material and distribute across the entire surface ofthe gas permeable material, and more preferably in a uniform surfacedensity. Skilled artisans are encouraged to recognize that through thepresent invention the word “horizontal” is inclusive of “substantially”horizontal, since under the weight of media the gas permeable materialcan move downward slightly in areas where it is not in direct contactwith a support. The intent of a substantially horizontal orientation isto allow cells to distribute across the gas permeable material.Preferably the substantially horizontal state of the growth surface issuch that it does not move out of plane by more than 20% of the surfacearea or the growth surface, more preferably 10%, even more preferably5%, and most preferably 2.5%.

When the animal cells to be cultured include adherent cells, the gaspermeable material is preferably hydrophilic and has an attachmentfriendly surface such as a plasma charged surface. Throughout thisdisclosure or any of the co-pending '814, '848, '597, '700, and '768specifications, skilled artisans are advised to recognize that the termgas permeable membrane is synonymous with gas permeable material and isnon limiting as skilled artisans are further advised to understand theword membrane is to be broadly defined as a gas permeable material ofany of the forms of material known by those of ordinary skill in the artto be commonly used for cell culture devices and cell culture methodsincluding those described in co-pending '814, '848, '597, '700, and'768. Throughout this disclosure the terms media and medium aresynonymous with liquid that contains any variety of substrates and/ornutrients that are used for the culture of animal cells. Preferably, allmaterials of the device that can be exposed to fluid associated with theculture process are compatible with cell culture (e.g. USP VI,non-cytotoxic, meet acceptable leachable and particulate standards,etc.). Also, to ensure one can determine if cells are being lost duringmedia removal, or assess the contents for any other reason, the cellculture and cell recovery device should preferably allow a visualassessment of the contents, as may be achieved by use of optically clearconstruction materials.

An aspect of one embodiment of the present invention can be found inco-pending '700 and its related drawing FIG. 22B, which is reproducedherein as FIG. 1A for illustrative purposes and for which item numbershave been altered from a 1000 series to a 100 series. In FIG. 1A, cellrecovery device 100 is shown in operation at the point where the culturehas been terminated and cells are about to be recovered. Cells 116reside upon growth surface 106, which forms the bottom of the device andwhich is comprised of gas permeable material of the characteristicspreviously described. Initial media volume 120A resides at an initialmedia height 121A, which is equal to the distance from the highest medialevel to the lowest media level, that is preferably beyond the 1.0 cmheight typical of static cell culture bags such as OriGen PermaLife™ andVueLife® bags. More preferably, initial media height 121A is at a heightbeyond 2.0 cm of medium. Although any height is possible, the optimalheight will depend upon the specifics of the cell culture application.For example, as described in co-pending '700, when expanding CAR T cellswithout need of medium exchange, an initial media height of 10 cm ispreferred if one seeks to allow a culture progress from a small numberof cells to a much greater number of cells without need of mediumexchange. Also, as media height increases, it makes it possible toremove a larger volume of media prior to collecting cells. We havediscovered that cells submerged under an unconventionally high level ofmedia do not easily become disbursed into the media as the media heightis reduced. Skilled artisans should be aware that removing a largevolume of media without disturbing cells can be very useful when seekingto perform media exchange (i.e. there is no requirement that cells beseparated from the media that is removed in order to be reintroducedinto the device and/or no requirement to split the culture to newdevices) or when terminating the culture to recover cells (i.e. cellseparation from the bulk of the media is performed in the device asopposed to use of cumbersome centrifugation equipment).

Unfortunately, it has been discovered that using standard closed systemfluid handling methods to take full advantage of the novel capacity toreduce media volume in devices such as those disclosed in co-pending'814, '848, '597, '700, and '768 can lead to cell loss and damage theculture devices, which for T cell therapy, adoptive immunotherapy,and/or adoptive cell therapy applications can be catastrophic.Collectively, FIG. 1B, FIG. 1C, and FIG. 1D illustrate an example of theproblems we have discovered in attempting to use traditional mediahandling tools and methods to reduce media volume prior to recoveringcells from G-Rex™ devices and other gas permeable devices such as thosedisclosed in co-pending '814, '848, '597, '700, and '768. FIG. 1B showsthe device described in FIG. 1A, after attaching waste receptacle 132 tomedia removal conduit 110. As a first step in reducing the amount ofinitial media volume 120A that cells 116 must be recovered from, aportion of initial media volume 120A is drawn from cell culture and cellrecovery device 100 by pumping it into waste receptacle 132 via mediaremoval conduit 110. A common method of pumping medium is by use ofperistaltic pump 134 to draw media out of the culture device. As shownin FIG. 1C, as a portion of the initial media volume is removed fromcell culture and cell recovery device 100, a pressure drop forms acrosssterile vent filter 128, placing internal volume 114 at a lower pressurerelative to ambient atmosphere and typically a vacuum quickly formswithin internal volume 114 of the device. Because growth surface 106 ispreferably comprised of a gas permeable material with gas transfercharacteristics that render it generally very thin and flimsy, when avacuum forms growth surface 106 is drawn out of its preferred horizontalplanar state very quickly to form a new, non-planar position as shown.Further, as rollers 136 of the peristaltic pump rotate, the gaps betweenthe rollers cause the vacuum within the device to pulse, thereby pulsingdistended growth surface 106, dislodging cells 116, and dispersing thedislodged cells throughout the medium. Subsequently, as shown in FIG.1D, as media continues to be withdrawn into waste receptacle 132, cells116 are also withdrawing in to waste receptacle 132. With this chain ofevents, the potential for a significant number of cells to be dispensedinto the waste receptacle is great. Since patient outcomes arecorrelated to the number of cells in a treatment dose, loss of theseprecious cells is potentially catastrophic. Also, even if cells are notlost, the growth surface has the potential to be drawn to the mediaremoval conduit making damage to the growth surface possible. Forexample a puncture of the growth surface can contaminate the culturerendering it unsuitable for patient use in addition to exposing those inthe cell manufacturing facility to potential biohazards. Even if thegrowth surface is not damaged by the media removal conduit, if it ispulled into the media removal conduit it can be blocked, preventing anyfurther removal of media and cells. In summary, drawing the growthsurface out of its planar and preferably horizontal state can lead tocell loss, the inability to remove cells, damage to the growth surfacedue to its distension, damage to the growth surface due to physicalcontact with the media removal conduit, contamination of the culture,and/or exposure of production workers to biohazards. Thus, methods ofcell recovery that avoid these pitfalls are needed.

In general, after performing cell culture and with cells havinggravitated to the bottom of the device, which is comprised of gaspermeable material, and when the device is still holding media, it isadvantageous if a volume of gas is moved into the cell culture and cellrecovery device in a manner that pressurizes the internal volume of thedevice. Preferably, a first volume of gas is moved into the device inorder to move a first volume of media from the device into a wastereceptacle. The step is preferable undertaken with the device orientedsuch that the growth surface upon which cells reside is oriented in ahorizontal plane. When this step is complete, a residual volume of mediaremains within the device and cells also remain in the device. A secondvolume of as is then moved into the device, thereby displacing theresidual volume of media and cells from the device and moving theresidual volume of media and cells into a cell collection vessel. In themanner, the ratio of the volume of media to number of cells is reduced.

FIG. 2A through FIG. 2E provide an illustrative example of an embodimentof the present invention that solves the problems shown in FIG. 1Bthrough FIG. 1D by pushing gas into the cell culture and cell recoverydevice to drive media and/or media and cells from the device underpressure, as opposed to drawing media from the device under vacuum. Thisapproach minimizes potential cell loss and/or damage to the device bypreventing a vacuum from forming, preventing upward distortion of thegrowth surface, and/or preventing pulsing the growth surface in and outof a planar state which can quickly dislodge cells. In FIG. 2A, across-sectional view of cell culture and cell recovery device 1000 isshown in a cell culture position and in a state of static cell culture.Cell culture and cell recovery device 1000 comprises an internal volume1014 bounded by an upper confine 1012 and a lower growth surface 1006,vent 1028, and media removal conduit 1010 with media removal opening1008. Media removal conduit clamp 1009 is in the closed position toretain media within internal volume 1014. Preferably upper confine 1012is adjoined to growth surface 1006 by sidewall(s) 1054 and morepreferably sidewall(s) 1054 are perpendicular to growth surface 1006 andare rigid to allow cell inoculum to gravity uniformly onto growthsurface 1006. An initial media volume 1020A is present in the device.Initial media volume 1020A resides within the confines of cell cultureand cell recovery device 1000 at initial media height 1021A, which isequal to the distance from the uppermost media level to the lowermostmedia level. Cells 1016 are also present, which have gravitated upongrowth surface 1006. Growth surface 1006 is preferably planar andoriented in a horizontal position during the culture process and iscomprised of non-porous, gas permeable, liquid impermeable material, andis hydrophobic in the case where non-adherent cells are to be cultured.Media removal opening 1008 resides a distance from growth surface 1006and as will be seen, the distance also constitutes a residual mediaheight 1021B. At some point in the cell culture process, performing amedia removal process that reduces initial media volume 1020A to asmaller residual media volume in order to either add fresh media orconcentrate the cells in the residual media volume becomes desirable.

To remove a portion of initial media volume 1020A, cell culture and cellrecovery device 1000 is oriented in a position such that growth surface1006 is at the bottom, and upper confine 1012 is at the top. Stateddifferently, cell culture and cell recovery device 1000 is oriented inits preferred static cell culture position. Cells 1016 reside on growthsurface 1006 at an initial cell density, which is the quantity of cells1016 divided by the initial media volume 1020A (e.g. cells/ml). Cells1016 also reside on growth surface 1006 at an initial surface density,which is the quantity of cells 1016 divided by the surface area ofgrowth surface 1006 upon which cells reside (e.g. cells/cm). A gasdelivery component is connected to vent 1028, which is preferablylocated at the top of the device as best shown in FIG. 2B. Regardless,of however the internal volume of the cell culture and cell recoverydevice is vented, it is preferably by way of a gamma radiation stablematerial capable of sterile filtering gas, such as a 0.2 micron ventfilter. In this example, the gas delivery component is diaphragm pump1050 which is connected to vent 1028 by gas conduit 1052. When the gasdelivery component is actively delivering gas (i.e. in this example whendiaphragm pump 1050 is actuated), gas is driven into gas conduit 1052,through vent 1028, and into cell culture and cell recovery device 1000.Most preferably vent 1028 is comprised of a material capable of ensuringgas moving into cell culture and cell recovery device 1000 is sterile.Vent 1028 is also preferably oriented in a position that minimizes theopportunity for condensation to accumulate upon its filtration surface.In this depiction, the filtration surface of vent 1028 is not parallelto growth surface 1006 and is instead oriented perpendicular to growthsurface 1006. As gas is delivered into cell culture and cell recoverydevice 1000 with media removal conduit clamp 1009 in the open position,a portion of initial media volume 1020A is displaced as it is driveninto the media removal opening 1008, out of cell culture and cellrecovery device 1000 by way of media removal conduit 1010, and into awaste receptacle, thereby leaving cells 1016 and a residual media volume1020B, which resides at a residual media height 1021B. The wastereceptacle need not be an enclosure and need not be physically attachedto media removal conduit 1010. For example it could instead be somethingas common as a laboratory sink, but preferably the waste receptacle isan enclosed container such as a bag and is connected in a sealed mannerto media removal conduit 1010 in order to isolate potential biohazardsin a closed system manner. Such an enclosed container is represented inFIG. 2A and FIG. 2B as enclosed waste receptacle 1032 which is attachedto media removal conduit 1010.

Preferably during this media removal process, growth surface 1006 isprevented from moving from its preferred horizontal position as gasenters cell culture and cell recovery device 1000 and the pressure ofinternal volume 1014 elevates. As will be described in the illustrativeembodiments, this can be accomplished by a growth surface support, whichholds the growth surface in a horizontal plane to prevent damage to thegrowth surface. Skilled artisans are advised to consult co-pending '814and '848 for further guidance related to the growth surface supportdesign.

After the media removal process is complete, fresh media can be added bycausing vent 1028 to be open to ambient atmosphere (e.g. disconnectingor venting gas conduit 1052) and adding fresh media via media removalconduit 1010 (or any other port that may present in the in the deviceand appropriately structured for that purpose). If there is no need ofadding fresh media into the device and the culture is to be terminated,cells 1016 can be removed by reorienting cell culture and cell recoverydevice 1000 from its preferred static cell culture position, as shown inFIG. 2C, to a cell removal position in which media removal opening 1008resides at a low point within internal volume 1014. Depending on thespecific characteristics of the cells and the growth surface, it may beuseful to agitate the residual media in the device to dislodge cellsfrom the growth surface and suspend them into the residual media priorto attempting to remove cells from the device. We have discovered thatnon-adherent cells such as T cells, even when the growth surface is ahydrophobic gas permeable material such as silicone, have a tendency toremain piled upon the growth surface even when the device is tilted tothe point where media no longer submerges the cells. Thus, to ensurecells are not left in the device when the residual media is removed,agitating the residual media to suspend cells within it prior towithdrawing residual media is preferred. Skilled artisans will recognizethere are many ways of suspending cells in the residual media. Forexample, one may simply impart motion to the device to swirl theresidual media about the device and over the growth surface and visuallydetermine when the cells have dislodged from the growth surface andmoved into the residual media. We have found that a cylindrical wallsurrounding a circular growth surface facilitates this approach well.However this step is accomplished, once cells are dislodged from thegrowth surface and dispersed into the residual media, collection ofcells can be undertaken by traditional methods of withdrawing the mediaand cells (such as by use of a peristaltic pump). However, withdrawingcells by drawing the media out of the device will cause the gaspermeable material to move from a planar position as a vacuum forms inthe device as a pressure drop across the vent occurs when the ventcomprises a sterile filter. Yet, so long as the integrity of the gaspermeable material is not breached as it moves from its horizontalposition into a new position, or the integrity of any other aspect ofthe cell culture and cell recovery device is not affected, this can bedone. However, a preferred method that does not put the gas permeablematerial or any other aspect of the cell culture and cell recoverydevice at risk of damage is to displace the residual media and cells bycausing gas to be delivered into the cell culture and cell recoverydevice.

FIG. 2D shows how this is accomplished. Cell culture and cell recoverydevice 1000 is oriented in the cell removal position, media removalconduit 1010 is preferably connected to cell collection receptacle 1040which replaces enclosed waste receptacle 1032, a gas delivery componentis connected to vent 1028, such as diaphragm pump 1050 which isconnected to vent 1028 by gas conduit 1052. When the gas deliverycomponent is actively delivering gas (i.e. in this example whendiaphragm pump 1050 is actuated), gas is delivered into gas conduit1052, through vent 1028, and into cell culture and cell recovery device1000. As gas is delivered into cell culture and cell recovery device1000, residual media volume 1020B, containing suspended cells 1016, isdisplaced as it is driven into media removal opening 1008 which emanatesfrom sidewall(s) 1054, out of cell culture and cell recovery device 1000by way of media removal conduit 1010, and into cell collectionreceptacle 1040. Preferably, the cell collection receptacle 1040 is anenclosed container with a suitable design for whatever downstreamprocesses it will be subjected to, such as centrifugation and/orcryopreservation. Preferably, in order to prevent damage to growthsurface 1006, a growth surface support is present and holds growthsurface 1006 in a planar state as gas enters cell culture and cellrecovery device 1000.

Skilled artisans are encouraged to recognize that increasing the heightat which the initial media volume resides above the bottom of the deviceis advantageous for a variety of reasons, including the ability toincrease the source of nutrients, increase the sink for cellular wasteproducts, and allow a greater portion of the initial media volume to beremoved without cell loss. Although media height is not limited,preferably the media height as determined from the lowest portion of themedia to the uppermost portion of the media when the growth surfaceand/or the uppermost surface of media is in a horizontal position ispreferably within a range of 1 cm to 25 cm, more preferably within arange of 1 cm to 20 cm, even more preferably within a range of 1 cm to15 cm, and even more preferably within a range of 2 cm to 11 cm.Although residual media height is not limited, preferably residual mediaheight is within a range of 0.2 cm to 2.0 cm, more preferably, within arange of 0.2 cm to 1.0 cm, and even more preferably within a range of0.2 cm to 0.5 cm as determined as determined from the lowest portion ofthe media to the uppermost portion of the media when the growth surfaceand/or the uppermost surface of media is in a horizontal position.

FIG. 3 shows yet another embodiment of the present invention in which across-sectional view of cell culture and cell recovery device 2000includes media removal opening 2008 located within media removal conduit2010. Media removal conduit does not access internal volume 2014 by wayof sidewall(s) 2054 and in this depiction moves through upper confine2012. Media can be collected as previously described when cell cultureand cell recovery device 2000 is oriented in the media collectionposition (i.e. the cell culture position) in which cells 2016 resideupon growth surface 2006. As shown in FIG. 3 , a portion of the originalmedia volume has been removed via media removal conduit 2010. Residualmedia 2020B resides at the height of media removal opening 2008. Cells2016 can be collected as previously described by simply reorienting cellculture and cell recovery device 2000 to the cell removal position, inwhich media removal opening 2008 resides at a low point of residualmedia volume 2020B. With cells 2016 suspended in residual media 2020B,they can be collected by removing residual media volume 2020B via mediaremoval conduit 2010.

Another embodiment of the present invention allows the opening of aconduit to alter its distance from the growth surface. In FIG. 4A, across-sectional view of a cell culture and cell recovery device 3000 isshown in a cell culture position and in a state of static cell culture.Cell culture and cell recovery device 3000 comprises an internal volume3014 bounded by an upper confine 3012, growth surface 3006, vent 3028,and media removal conduit 3010 with media removal opening 3008.Preferably upper confine 3012 is adjoined to growth surface 3006 bysidewall(s) 3054 and more preferably sidewall(s) 3054 are perpendicularto growth surface 3006 during the culture process.

Media can be removed via media removal opening 3008 in the mannerpreviously described, preferably when cell culture and cell recoverydevice 3000 resides in a position in which planar growth surface 3006resides in a horizontal position and cells reside upon the growthsurface, not suspended in the media (i.e. not distributed within themedia). After the media removal process is complete, fresh media can beadded by causing vent 3028 to be open to ambient atmosphere (e.g.disconnecting or venting the gas conduit) and adding fresh media viamedia removal conduit 3010 (or any other port that may present in the inthe device and appropriately structured for that purpose). If there isno need of adding fresh media into the device and the culture is to beterminated, cell removal can be undertaken. By configuring the cellculture and cell recovery device to include means for altering thedistance between media removal opening 3008 and growth surface 3006, thelocation of media removal opening 3008 can be altered from a mediaremoval position to a cell removal position. In so doing, not only canthe media removal opening act to reduce the media volume withoutremoving cells, as described previously, it can also remove residualmedia and cells after media volume has been reduced. In this manner,just one port can be used to concentrate the cells in residual media andalso remove the concentrated cells. FIG. 4B shows one example of how toaccomplish this. In this example, sidewall(s) 3054 provide structureconnecting the lower boundary of the internal volume of the cell cultureand cell recovery device to the upper confine. In this depiction, growthsurface 3006 is the lower boundary. Sidewall(s) 3054 of cell culture andcell recovery device 3000 includes means to adjust the distance betweenmedia removal opening 3008 and growth surface 3006. Skilled artisans areencouraged to recognize there are many way of accomplishing this. Forexample, sidewall(s) 3054 can include flexible material such as siliconeand be bellowed to allow the height of sidewall(s) 3054 to be altered.When the height of sidewall(s) 3054 is altered to move media removalopening 3008 from a media removal position to a cell removal position asshown, the distance between media removal opening 3008 and growthsurface 3006 is reduced and media removal opening 3008 is placed in acell removal position where it is in proximity of growth surface 3006and is able to allow passage of residual media and cells. Stateddifferently, when cell culture and cell recovery device 3000 is in themedia removal position, the distance between media removal opening 3008and growth surface 3006, which in this depiction is the lower boundaryupon which media can reside when the device is operating in a state ofstatic cell culture, exceeds the distance between media removal opening3008 and growth surface 3006 when in the cell removal position.Conversely, when cell culture and cell recovery device 3000 is in thecell recovery position, the distance between media removal opening 3008and growth surface 3006 is less than the distance between media removalopening 3008 and growth surface 3006 when cell culture and cell recoverydevice 3000 is in the media removal position.

Skilled artisans are advised the ways to adjust the distance between themedia removal opening and the growth surface are many and varied. Forexample, there are numerous ways to make the walls of the device capableof collapsing, including making them out of a flexible material, in apiston like manner where one section of the wall slides past the otherin a liquid tight manner, and the like. In those cases, the distancebetween the upper confine of the cell culture and cell recovery deviceand the growth surface will be reduced as the media removal opening isaltered from a media removal position to a cell removal position.However, skilled artisans are advised to recognize that altering thedistance between the upper confine of the cell culture and cell recoverydevice and the growth surface is not the only way to move the mediaremoval opening from a media removal position to a cell removalposition.

FIG. 5A and FIG. 5B show a cross-sectional view of cell culture and cellrecovery device 4000 configured to adjust the distance between mediaremoval opening 4008 and growth surface 4006. Flexible shroud 4001 formsa seal against media removal conduit 4010 and upper confine 4012. Mediaremoval conduit 4010 has a sealed joint, which in this illustrativeexample is comprised of female luer fitting 4011 that interfaces withmale luer fitting 4013, thereby allowing collection vessels to be easilyattached to media removal conduit 4010. Skilled artisans are encouragedto recognize there are many ways to make such a connection includingsterile tubing welds. To alter the distance between media removalopening 4008 and growth surface 4006, the height of flexible shroud 4001is altered. FIG. 5B shows the height of flexible shroud 4001 after aforce has been applied to its upper surface in order to diminish itsheight (i.e. compressing the bellows in the direction of growth surface4006), thereby moving media removal opening 4008 in the direction ofgrowth surface 4006 and reducing the distance between media removalopening 4008 and growth surface 4006. Skilled artisans are encouraged torecognize that a variety of distances between media removal opening 4008and growth surface 4006 become possible without putting cell culture andcell recovery device 4000 at risk of contamination. Preferably, duringcell recovery, media removal opening 4008 is adjacent to growth surface4006, and cell culture and cell recovery device 4000 is in the cellculture position (i.e. growth surface 4006 is in a horizontal position).This facilitates automated processing in a manner that does not requirethe cell culture and cell recovery device to be rotated from its cellculture position to remove media and cell contents of the cell cultureand cell recovery device.

Prior to the collection of cells, it may be helpful to agitate thedevice to dislodge cells from the growth surface and suspend them intothe media. We have discovered that non-adherent cells such as T cells,even when the growth surface is a hydrophobic membrane such as silicone,have a tendency to remain piled upon the growth surface when the deviceis tilted. Thus, to ensure cells are not left in the device when theresidual media is removed, agitating the device is preferred. Forexample, one may simply swirl the residual media about and visuallydetermine the cells have dislodged from the growth surface and movedinto the residual media. Once cells are dislodged from the growthsurface, collection of cells can be accomplished by traditional methodsof withdrawing the media and cells (such as by use of a peristalticpump). Withdrawing cells by drawing the media out of the device willcause the gas permeable membrane to move from a horizontal position as avacuum forms in the device. So long as the integrity of the membrane isnot breached, this can be done. However, a preferred method that doesnot put the membrane at risk of damage, is to use the gas displacementmethod previously described to recover the cells.

FIG. 5C shows cell culture and cell recovery device 4000 configured tofurther assist automated cell collection by eliminating the need to tiltthe device to place the conduit opening being used to collect cells atthe lowest position within the device. When growth surface 4006 residesin the preferred horizontal plane for cell culture, cell collectionpocket 4007 resides below the horizontal plane of growth surface 4006.Media removal opening 4008 resides within cell collection pocket 4007.In this position, media removal opening 4008 resides at the low point ofinternal volume 4014 (i.e. the lowermost location of media when thedevice is oriented for a state of static cell culture), facilitatingrecovery of media and cells. This feature can be present in any cellculture and cell recovery device embodiment.

In general, when media removal opening is in the media removal position,it preferably does not have the ability to remove all contents of thecell culture and cell recovery device. This ensures at least a portionof media and the majority of cells remain in the device even if the gasdelivery component becomes defective, or fails to be stopped fromdelivering gas, and continues to deliver gas into the device after allthe media that can possibly move through the media removal opening hasdone so.

If one seeks to avoid designing the cell culture and cell recoverydevice in a manner that alters the distance between the media removalopening and the growth surface, or requires a large angle of rotation tocollect cells, it is easy to create a distinct media removal opening anda distinct cell removal opening and apply the process previouslydescribed to simplify downstream processing without fear of cell loss ordamage to the growth surface and/or the gas permeable material. FIG. 6A,FIG. 6B, FIG. 6C, and FIG. 6D provide such an example. A cross-sectionalview of a cell culture and cell recovery device 5000 is shown a staticcell culture position comprising an internal volume 5014 bounded by anupper confine 5012, a lower boundary which in this depiction growthsurface 5006, vent 5028, media removal conduit 5010 with media removalopening 5008, and cell removal conduit 5004 with cell removal opening5002. Preferably upper confine 5012 is adjoined to the lower boundary bysidewall(s) 5054 and more preferably sidewall(s) 5054 are perpendicularto the growth surface. An initial media volume 5020A is present in thedevice. Initial media volume 5020A resides at an initial media height5021A, which is equal to the distance from the highest media level tothe lowest media level. In use, cells 5016 reside on growth surface5006. In general, skilled artisans should be aware that non-adherentcells (i.e. also called suspension cells) may not all touch the growthsurface since they will be able to pile upon each other with the lowermost cells in actual physical contact with the growth surface. Growthsurface 5006 is preferably oriented in a horizontal position during theculture process, is preferably comprised of non-porous liquidimpermeable material, and is preferably hydrophobic in the case wherenon-adherent cells are to be cultured. Cells 5016 are at an initial celldensity, which is the quantity of cells 5016 divided by the initialmedia volume 5020A (e.g. cells/ml), and cells 5016 are also at aninitial surface density, which is the quantity of cells 5016 divided bythe surface area of growth surface 5006 upon which cells reside (e.g.cells/cm). Again, in the case of non-adherent cells this includes thetotal number of cells that have gravitated to a state of rest and whichare prevented from further gravitation by the growth surface. Thisincludes those cells that have come to rest upon other cells, some ofwhich are not in direct contact with the growth surface. Media removalopening 5008 resides a distance from growth surface 5006 and as will beseen, the distance also constitutes residual media height 5021B.

At some point in the cell culture process, performing a media removalprocess that reduces initial media volume 5020A to a smaller residualmedia volume in order to either add fresh media or concentrate the cellsin the residual media volume becomes desirable. To remove a portion ofinitial media volume 5020A, a gas delivery component is connected tovent 5028 as best shown in FIG. 6B. In this example, the gas deliverycomponent is diaphragm pump 5050 which is connected to vent 5028 by gasconduit 5052. When diaphragm pump 5050 is actuated, it drives gas intogas conduit 5052, through vent 5028, and into cell culture and cellrecovery device 5000. As gas is driven into internal volume 5014 of cellculture and cell recovery device 5000, a portion of initial media volume5020A is displaced and is driven into media removal opening 5008,through media removal conduit 5010 and out of cell culture and cellrecovery device 5000 into a waste receptacle, leaving residual mediavolume 5020B and cells 5016 as shown in FIG. 6C. Residual media volume5020B resides at residual media height 5021B, defined as the distancebetween the uppermost residual media location and the lowermost residualmedia location. Cells 5016 are at a residual cell density, which is thequantity of cells 5016 divided by residual media volume 5020B (e.g.cells/ml), and cells 5016 are also at residual surface density 5017B,which is the quantity of cells 5016 divided by the surface area ofgrowth surface 5006 upon which cells reside (e.g. cells/cm²). The wastereceptacle need not be an enclosure and need not be physically attachedto media removal conduit 5010. For example it could instead be somethingas common as a laboratory sink, but preferably the waste receptacle isan enclosed container such as a bag or centrifuge tube and is connectedin a sealed manner to media removal conduit 5010 in order to isolatepotential biohazards and/or maintain sterility in a closed systemmanner. Such an enclosed container is represented in FIG. 6C as enclosedwaste receptacle 5032 which is attached to media removal conduit 5010.As a portion of initial media volume 5020A is being removed from cellculture and cell recovery device 5000, the volume of media in wastereceptacle 5032 is initial media volume 5020A less the portion ofinitial media volume 5020A within media removal conduit 5010 (if any).Preferably during this media removal process, growth surface 5006 isprevented from moving in the opposite direction from upper confine 5012without constraint as gas enters cell culture and cell recovery device5000 and the pressure of internal volume 5014 elevates. As shown in FIG.6C, this is accomplished by growth surface support 5018, which makescontact with growth surface 5006 and holds growth surface 5006 in asubstantially planar position to prevent damage to the growth surface.The term substantially has been previously described as it relates to ahorizontal position and this also applies to a planar position. Skilledartisans are advised to consult co-pending '814 and '848 for furtherguidance related to the growth surface support design.

Referring to FIG. 6D, to recover cells 5016, a further step isundertaken in which cell collection receptacle 5040 is attached to thecell removal conduit 5004 of the cell culture and cell recovery device5000 and media removal conduit 5010 is blocked from fluid flow by closedmedia removal conduit clamp 5009. Blocking media removal conduit 5010from fluid flow may be as simple as use of a hemostat, or may beautomated as will be described further into this disclosure. Aspreviously described, prior to the collection of cells it may be helpfulto agitate the residual media within the device to dislodge cells fromthe growth surface and suspend them into the residual media. Althoughcollection of cells can be accomplished by traditional methods ofwithdrawing the media and cells (such as by use of a peristaltic pump),the preferred method is to prevent the growth surface and/or gaspermeable material from moving toward the upper confine and distortingin shape during this process by use of the positive pressure the gasdisplacement method provides. As shown in FIG. 6D a gas deliverycomponent is connected to vent 5028 by way of gas conduit 5052. In thisexample, the gas delivery component is diaphragm pump 5050. Whendiaphragm pump 5050 is actuated, it drives gas into gas conduit 5052,through vent 5028, and into the cell culture and cell recovery device5000. As gas is driven into cell culture and cell recovery device 5000,residual media 5020B and cells 5016 are displaced and driven into cellremoval opening 5002, through cell removal conduit 5004, and into cellcollection receptacle 5040. Cell collection receptacle 5040 can then beremoved, preferably in a sterile manner such as by way of a steriletubing weld, for subsequent processing of cells 5016.

It is possible a rinse of a portion of the internal volume cell cultureand cell recovery device may be desired to collect any cells that mayremain in the device after the initial cell removal process. Forexample, this can be accomplished without need to add a source of liquidthat was not present in the device at the onset of the cell removalprocess. One could do so by simply using the media residing in theenclosed waste receptacle as the rinse material. To do so, the mediaremoval clamp would be opened, the vent would be opened, and the wastereceptacle would be pressurized such as by simply elevating it towhatever height is needed to allow media to flow back into the device.This can be assisted by squeezing the waste receptacle if it isflexible, such as a bag, to initiate flow into the device. When a userdetermines an adequate amount of media has been returned to the device,the media removal conduit clamp can be closed and the cell removalprocess can be repeated

Although the described process of using air to displace media and/orcells can be undertaken in a simple manner such as by opening andclosing conduits with a hemostat and turning the gas delivery componenton and off by visual assessment of the process, automating aspects ofthe process can be beneficial in the case where many cell preparationsare desired. One of the factors to consider is the prospect of the gasdelivery component continuing to deliver gas to the device after mediaand/or cells have been driven into their respective receptacles. In thiscase, gas would be driven into the receptacles and the receptacles wouldbe pressurized, potentially causing them to breach their seal.Furthermore, gas could cause damage to the cells as the surface tensionof bubbles and gas/cell contact may disrupt the integrity of the cellmembranes. Another factor to consider is the pressure that can buildwithin the cell culture and cell recovery device itself when the gasdelivery component is not turned off after media and/or cells are drivento their respective receptacles.

FIG. 7 shows a schematic of an illustrative embodiment of systemdesigned to resolve these problems. Skilled artisans are encouraged torecognize this illustrative embodiment is compatible with a cell cultureand cell recovery device configured with just one media removal conduitas for example shown in FIG. 3 , an adjustable media removal conduit, orseparate media and cell removal conduits. In general, the automationsystem preferably recognizes the point in the process when media exitingthe cell culture and cell recovery device via a media removal conduit ora cell removal conduit is replaced by gas. Referring again to FIG. 7 ,fluid detection components 6044A and 6044B are capable of recognizingthe point in the process when media exiting the cell culture and cellrecovery device via a media removal conduit or a cell removal conduit isreplaced by gas and are in proximity of media removal conduit 6010 andcell removal conduit 6004. Stated differently, the fluid detectioncomponents are able to determine if liquid or gas resides in theconduits. First flow control component 6046A acts to open or close themedia removal conduit 6010 and second flow control component 6046B actsto open or close cell removal conduit 6004. In this example,electronically actuated pinch clamps act as the flow control componentsand the conduits are made of flexible tubing. Cell culture and cellrecovery device 6000 is preferably a static cell culture device in itscell culture position (i.e. media is not subject to forced mixing andthe growth surface is below the upper confine and is preferably in thehorizontal position). Media 6020 resides at a first media volume and ata first media height within cell culture and cell recovery device 6000and cells 6016 have settled upon growth surface 6006 due to gravity.Growth surface 6006 is preferably held in a horizontal plane by growthsurface support 6018. The distance from media removal opening 6008 togrowth surface 6006 exceeds the distance from cell removal opening 6002to growth surface 6006. Preferably, the cell removal conduit is incontact with a sidewall, is in contact with the growth surface, ispositioned along the edge where sidewall(s) 6054 interface(s) with thegrowth surface, or in a pocket within the growth surface as previouslydescribed, thereby positioning the cell removal opening for maximum cellrecovery. Gas conduit 6052 connects vent 6028 to gas delivery component6050. Media removal conduit 6010 is connected to waste receptacle 6032and cell removal conduit 6004 is attached to cell collection receptacle6040. A software algorithm is able to deliver the electronic signalsneeded to perform the various tasks needed to collect cells in a highlyconcentrated state. To reduce the volume of media 6020, flow controlcomponent 6046A is placed in a state that does allow fluid flow throughmedia removal conduit 6010 and flow control component 6046B is placed ina state that does not allow fluid flow through cell removal conduit6004, the gas delivery component is actuated and gas enters cell cultureand cell recovery device 6000 via vent 6028, pressurizing internalvolume 6014 and driving media 6020 into media removal opening 6008,through media removal conduit 6010, and into waste receptacle 6032. Whenthe height of media 6020 has reached a level that is just a minisculedistance below at the height of media removal opening 6008, gas entersmedia removal opening 6008, is driven through media removal conduit6010, and is detected by fluid detection component 6044A. Fluiddetection component 6044A sends a signal notifying flow controlcomponent 6046A to stop fluid flow to waste receptacle 6032. Preferablygas delivery component 6038 is simultaneously terminated from deliveringgas and/or opens pressure relief valve 6056. Although this step isoptional, the advantage is to minimize pressure build up in cell cultureand cell recovery device 6000 and skilled artisans are encouraged torecognize there are numerous ways to achieve that objective includinguse of a gas delivery component that cannot deliver gas after a specificpressure threshold is reached, use of a pressure relief valve, and thelike. Once media 6020 flow to waste receptacle 6032 is terminated, theoperator can then make a determination as to whether or not to agitatethe residual amount of media in order to dislodge cells 6016 from growthsurface 6006. Skilled artisans are encouraged to recognize that a robustsystem will allow the operator to override the automation and stop themedia flow to the waste receptacle on command (e.g. as may be needed ifcells are being lost into the waste receptacle).

Once the operator has determined that cells 6016 are in a suitable stateof suspension within the residual amount of media 6020 and/or the cellrecovery process should commence, the user presses a button and theprocess resumes. Internal volume 6014 is pressurized and flow controlcomponent 6046B is actuated to allow fluid flow to cell collectionreceptacle 6040. Cell culture and cell recovery device 6000 may or maynot be oriented into a position to allow all the media and cells tocollect at a low point at this time, depending on operator preferenceand whether or not the cell culture device is designed to place the cellremoval conduit in a position of maximum cell recovery when the deviceis oriented in the static cell culture position. Skilled artisans willrecognize the steps of agitation to dislodge cells and orienting thedevice into a position of maximum cell recovery can also be automated.When in the static cell culture position, maximum cell recovery can beobtained when cell removal opening 6002 is located below the height ofgrowth surface 6006. For example, by a pocket in the growth surface aspreviously described, a moat such as a relieved area around theperimeter of the growth surface where the bottom of the moat is lowerthan the growth surface, or any feature that could act to allow media toreside at a height below the growth surface and become a collectionlocation for the cell removal opening. At some point in the automatedcell removal process, gas enters cell removal opening 6002, travelsthrough cell removal conduit 6004, and is detected by fluid detectioncomponent 6044B. At that point, fluid detection component 6044B sends asignal causing flow control component 6046B to terminate fluid flow tocell collection receptacle 6040. At this point, receptacle 6040 can beremoved, preferably in a sterile manner by use of a sterile tubingwelder. However, if the operator determines an additional rinse of thegrowth membrane can be useful in collecting more cells, such that anythat may have remained within the device, that process becomes easilypossible. Flow control component 6046A can be placed in a state wherefluid flow through media removal conduit 6010 is not blocked, butinstead is open, and waste receptacle 6032 is simply elevated orsqueezed (preferably the waste receptacle is a bag) in order to drivemedia 6020 from waste receptacle 6032 into cell culture and cellrecovery device 6000. Preferably vent 6028 is open to atmosphere duringthis step. Once an appropriate volume of media 6020 is delivered backinto cell culture and cell recovery device 6000, the cell recoveryprocess can be repeated. This step of adding and removing media can berepeated as necessary to collect as many cells as the operator deemsappropriate.

The method of using a device of the embodiment shown in FIG. 7 would besuch that the gas delivery component is connected to a filter that isconnected to the gas permeable cell culture and cell recovery devicecontaining media and cells wherein the cell culture device includes amedia removal conduit, connecting the first fluid detection component tothe media removal conduit, connecting the first fluid flow controlcomponent to the media removal conduit, initiating gas delivery from gasdelivery component, whereby initiating can be use of any process orequipment that causes gas to move into the cell culture device andwherein the gas displaces media from the cell culture device into amedia collection vessel connected to the media removal conduit. Thefirst fluid detection component determines when the fluid that is movingthrough the media removal conduit has changed from liquid to gas and thefirst fluid detection component sends a signal to the first fluid flowcontrol component and upon receiving the signal the first flow controlcomponent terminates the flow of fluid through the media removalconduit. After the first flow control component has terminated the flowof fluid through the media removal conduit, an option of performingagitation of the media is to dislodge cells from the growth surface intothe residual media can be undertaken. The cell culture device can alsobe oriented into a new position wherein media is in contact with said acell removal conduit. However, if the growth surface remains in thehorizontal position and the cell removal opening of the cell removalconduit is in contact or proximity of the growth surface, such a step ofreorienting the device need not be undertaken. A cell collection vesselis connected to the cell removal conduit, the second flow controlcomponent opens the flow of fluid through the cell removal conduit, gasfrom the gas delivery component moves into the cell culture device,media and cells move through the cell removal conduit into a cellcollection vessel, the second fluid detection component determines whenthe fluid that is moving through the cell removal conduit has changedfrom liquid to gas and sends a signal to a second fluid flow controlcomponent. Upon receiving the signal, the second flow control componentterminates the flow of fluid through the cell removal conduit.

Skilled artisans are encouraged to recognize that the device describedin the illustrative embodiment of FIG. 7 can be simplified if it were tointerface with a cell culture and cell recovery device of the type withjust one media conduit that acts to reduce media volume and acts toremove residual media volume and cells. Such a device is described infurther for example detail in FIGS. 2A-2D, 3, 4A-4B, 5A-5B and relatedtext. Thus, the device can be simplified in include a gas deliverycomponent that is capable of connecting to a filter that is connected toa gas permeable cell culture device, the gas delivery component capableof delivering gas into said gas permeable cell culture device by way ofsaid filter, a first fluid detection component that is capable ofdetermining when the type of fluid moving within a media removal conduitthat is connected to a gas permeable cell culture device changes fromliquid to gas, the component capable of sending a signal to a firstfluid flow control component that is capable of terminating fluid flowthrough said media removal conduit. After the first flow controlcomponent has terminated the flow of fluid through the media removalconduit, if need be the cell culture device is oriented into a newposition wherein media is in contact with the media removal conduit, acell collection vessel is connected to the media removal conduit, firstflow control component opens the flow of fluid through the media removalconduit, gas from said gas delivery component moves into said cellculture device, media and cells move through the media removal conduitinto the cell collection vessel, the first fluid detection componentdetermines when the fluid that is moving through the media removalconduit has changed from liquid to gas and sends a signal to the firstfluid flow control component. Upon receiving the signal the first flowcontrol component terminates the flow of fluid through said mediaremoval conduit.

Skilled artisans will recognize that it is beneficial for cells to moveout of the cell removal conduit and into the cell collection receptacleto the maximum extent practical. Thus, when automation is used in themanner shown in FIG. 7 , by positioning the fluid detection componentdownstream from the flow control mechanism and as near to the receptacleas possible, the amount of media in the conduit at the time the flowcontrol mechanism terminates flow will be limited to that between theposition of the fluid detection component and the cell collectionreceptacle. Also, minimizing tubing inner diameter can further reducethe number of cells in the conduit. Alternatively, after gas is detectedin a conduit, a small amount of additional gas can be driven throughconduit(s) to ensure liquid is not trapped in the conduit(s) when theprocess is complete. This may be useful to ensure all cells are movedinto the cell collection receptacle and/or allow sterile tubing splicingwithout liquid in the conduits. Alternatively, gas can be driven throughthe conduit to ensure liquid is not trapped in the conduit when theprocess is complete.

Skilled artisans are encouraged to recognize that the use of flowcontrol components and fluid detection components are a convenience, butthe cell recovery process can be undertaken manually. In a manual cellrecovery process, an operator cold clamp flow through the cell removalconduit and open flow through the media removal conduit, and then coulduse a syringe to inject gas into the device until the gas is moved intothe media removal conduit. Then the media can be agitated out of a stateof quiescence as needed to dislodge cells from the growth surface. Withthe media removal conduit closed and the cell removal conduit open, gasis added into the device thereby driving cells and residual media out ofthe device.

Although the use of gas to displace media in order to prevent distortionof the growth surface and/or gas permeable material is an elegantapproach, the device can be configured to minimize distortion growthsurface and/or gas permeable material if one desires to withdraw mediaand/or cells from the device as opposed to displacement by gas aspreviously described. Such an approach can take place with the internalvolume of the device at a pressure that is less than the pressureexternal to the device, thereby creating a pressure differential acrossthe walls of the device. One approach to preventing distortion of thegrowth surface out of a planar state under such conditions is tophysically attach the gas permeable material to a component (such as forexample the growth surface support component). FIG. 8 and FIG. 9 providetwo such examples. For simplicity, the entire cell culture and cellrecovery device is not shown. In FIG. 8 , growth surface 6006 includestabs 6058 emanating from its lower surface. Tabs 6058 interlock withmating features on growth surface support 6018. Vertical supports 6018Aproject from the base of growth surface support 6018 to hold growthsurface 6006 in a horizontal position during static cell culture.Preferably, growth surface 6006 is comprised of a gas permeablematerial. In the case of silicone, the membrane can be fabricated byliquid injection molding and tabs 6058 can be molded into the membrane.FIG. 9 shows another example of maintaining the growth surface in asubstantial planar state when pressure external to the growth surfaceexceeds that of the internal volume of the cell culture and cellrecovery device. In this illustrative example, growth surface 7006 iscomprised of silicone, which is over-molded onto grid 7060 (i.e.attached to the grid) which is attached to growth surface support 7018.Gas access openings 7018B allow passive gas exchange of the culture.Stated differently, ambient gas makes contact with the growth surfacewithout need of being forced to do so. Thus, growth surface 7006 is heldin place via the contact points with growth surface support 7018 evenwhen pressure external to the growth surface exceeds that of theinternal volume of the cell culture and cell recovery device.

Another way of maintaining the growth surface in a substantially planarstate when media and/or fluid is drawn from the device is to balance thepressure external to the growth surface with that of the internal volumeof the cell culture and cell recovery device. FIG. 10 shows anillustrative example of how that can be accomplished. Cell growth andcell recovery device 8000 includes growth surface support 8018. Growthsurface support 8018 includes gas access opening(s) 8021. Gas accessopening(s) allows passive movement of ambient gas to and from growthsurface 8006, which is preferably liquid impermeable, gas permeable,non-porous, and residing in a substantially horizontal plane during cellculture. Stated differently, by passive movement the ambient gas makescontact with the gas permeable growth surface without any mechanism tophysically force gas into contact with the gas permeable material. Mediaremoval conduit 8010 is attached to waste receptacle 8032 and interfaceswith a peristaltic pump 8030 in a manner such that peristaltic pump 8030is able to draw media 8020 out of cell growth and cell recovery device8000. During media removal, the ability of gas access opening(s) 8021 tocommunicate with ambient gas is minimized and preferably eliminated. Inthis depiction, liquid removal adapter 8027 is temporarily attachedwhile the volume of media is reduced and mates with growth surfacesupport 8018 and includes a pressure balance conduit 8033A, whichfurther includes pressure balance conduit interface 8029, as for examplemay be a luer interface. Pressure balance conduit 8033A can mate topressure balance conduit 8033B. Pressure balance conduit 8033B can alsobe attached to media removal conduit 8010 so that peristaltic pump 8030,when actuated, can draw gas from the space between liquid removaladapter 8027 and growth surface support 8018, in effect placing gasspace 8019 at a reduced pressure relative to ambient gas. Preferably, ifpressure balance conduit 8033B is attached to media removal conduit8010, pressure balance conduit 8033B optionally includes check valve8034 and/or pressure balance conduit filter 8035, capable of allowinggas to move to waste receptacle and preventing movement of media 8020into it while simultaneously maintaining sterility of the device and itsfluid connections. Skilled artisans are encouraged to recognize thatpressure balance conduit 8033B need not be attached to media removalconduit 8010, but can instead be a separate conduit interfaces with aperistaltic pump. In this case, preferably a multichannel peristalticpump is used that is connected to the media removal conduit and thepressure balance conduit. In such configurations, peristaltic pump 8030draws gas from below growth surface 8006, pulling a vacuum on growthsurface 8006 that counter balances the vacuum being created within cellgrowth and cell recovery device 8000, thereby holding growth surface8006 in a substantially horizontal state, or at a minimum lessening inthe distortion experienced by growth surface 8006. Skilled artisans areencouraged to recognize that any apparatus that draws gas out of thepressure balance conduit can achieve the purpose of preventingpotentially damaging distortion to the growth surface as media isremoved.

Growth surface support 8018 is designed to interface with liquid removaladaptor 8027 and growth surface support 8018 is designed to interfacewith cell culture and cell culture recovery device 8000 in a manner thatallows gas to be removed from gas space 8019 at a rate that allows thepressure adjacent and external to the growth surface to be equal or lessthan the pressure of internal volume 8014. Skilled artisans are advisedto recognize that the interfaces need not be an air tight seal so longas it exerts control over gas space 8019 pressure. Preferably however,seals are provided. Skilled artisans should also be advices that liquidremoval adapter 8027 need not be required to achieve the objective ofmaintaining the growth surface in a substantially horizontal state asmedia is drawn from the cell culture and cell recovery device. Forexample, pressure balance conduit 8033B can interface directly with gasaccess opening(s) 8021. In any event, if used, the liquid removaladaptor is preferably easily connected and disconnected from the growthsurface support such that when disconnected, ambient gas can makepassive contact with the growth surface without need of any mechanismsor processes to force gas. Skilled artisans should be aware that thegrowth surface support need not be permanently attached to the cellculture and cell recovery device.

FIG. 11 shows another embodiment of a cell culture and cell recoverydevice adapted to allow media to be withdrawn from the device withsubstantially little distortion of the growth surface. Cell growth andcell recovery device 9000 includes growth surface support 9018. Growthsurface support 9018 includes gas access opening(s) 9021. Gas accessopening(s) 9021 allow passive movement of ambient gas to and from growthsurface 9006, which is preferably gas permeable, liquid impermeable, andresiding in a horizontal plane during cell culture. Gas access openingfilter 9036 prevents contaminants from accessing gas access opening(s)9021. Skilled artisans are advised that the material selected for thegas access opening filter can be any such that it acts as a sterilebarrier and allows passive gas access to the growth surface at a ratethat allows adequate oxygenation of the culture. Preferably, it is aporous material with pore sizes of less than 0.45 microns and morepreferably less than 0.22 microns. Media removal conduit 9010 isattached to waste receptacle 9032 and interfaces with and peristalticpump 9030 in a manner such that peristaltic pump 9030 is able to drawmedia 9020 out of cell growth and cell recovery device 9000.

Pressure balance conduit 9033 links media removal conduit 9010 to gasspace 9019 and may optionally contain check valve 9034 (to prevent mediafrom potentially entering the gas space) and/or pressure balance conduitfilter 9036 (to prevent contaminants or biohazards from potentiallyentering the gas space and then potentially crossing the gas accessopening filter). During media removal, the peristaltic pump rate is suchthat a pressure in gas space 9019 (i.e. the space between growth surface9006 and gas access opening filter 9036) is less than or equal to thepressure of internal volume 9014, thereby minimizing and/or preventingmovement of growth surface 9006 towards upper confine 9012. Preferably,the pressure balance conduit is a flexible tube that mates to a tubeemanating from an accessing opening to the gas space, and a steriletubing weld is used to create the mate.

Yet another embodiment for a cell culture and cell recovery device andprocess is shown in FIGS. 12A and 12B. In this embodiment, the internalvolume of the cell culture and cell recovery device is placed at apressure that is less than that of the ambient environment as media ispulled out of the cell culture and cell recovery device. FIG. 12A showscell culture and cell recovery device 10000 shown in a state of staticcell culture. Cells 10016 have gravitationally settled onto gaspermeable, liquid impermeable, growth surface 10006. In FIG. 12B,ambient gas restrictor 10018 is in contact with cell culture and cellrecovery device 10000. Ambient gas restrictor 10018 acts to limitambient gas contact with growth surface 10006 in order to limit thepressure imbalance across growth surface 10006 when liquid is pulledfrom cell culture and cell recovery device 10000. In this example,peristaltic pump 10030 acts to draw media 10020 out of media removalconduit 10010. As media is drawn out of the cell culture and cellrecovery device, a pressure drop becomes present when gas is restrictedfrom entering the device by vent filter 10028. Despite the pressure ofthe internal volume of cell culture and cell recovery device 10000having been reduced relative to the ambient environment, growth surface10006 is prevented from experiencing the relative pressure imbalance byambient gas restrictor 10100. Ambient gas restrictor 10100 need not makea gas tight connection to cell culture and cell recovery device 10000,but that is preferable if one wishes to limit substantial movement ofthe growth surface toward upper confine 10012. The process of reducingmedia volume prior to cell recovery can be carried out as previouslydescribed using one or more conduits. Preferable, if present, growthsurface support 10018 is designed to minimize gas within it.

Yet another embodiment for a cell culture and cell recovery device andprocess is shown in FIGS. 13A, 13B, and 13C. In this approach, a fixedand predetermined volume of gas is injected into the cell culture andcell recovery device. Although the source of the injected gas in thisillustrative embodiment is a position, skilled artisans are encouragedto recognize that the source can be any mechanism(s) able to dispense aknown volume of gas, such as a bellows. As shown in FIG. 13A, cellculture and cell recovery device 11000 is shown in a state of staticcell culture and includes initial media volume 11020A and cells 11016.Piston 11100 is connected to cell culture and cell recovery device 11000by way of vent filter 11028. As shown in FIG. 3B, when waste media is tobe removed, piston head 11150 moves a first predetermined distance todrive a volume of gas into cell culture and cell recovery device 11000.The volume of gas drives an equal volume of media out of media removalconduit 11010, which is open to a waste receptacle (not shown forclarity). Upon completion of this step, media removal conduit 11010 isclosed to the waste receptacle and a residual media volume 11020Bremains in cell culture and cell recovery device 11000 along with cells11016. As shown in FIG. 13C, when cells 11016 and residual media 11020Bare to be removed, piston 11100 moves a second predetermined distance todispense an additional volume of gas into cell culture and cell recoverydevice 11000, thereby driving cells 11016 and the residual media volume11020B through an open cell removal conduit 11004 and into a cellcollection receptacle (not shown for clarity).

Those skilled in the art will recognize that numerous modifications canbe made thereof without departing from the spirit. Therefore, it is notintended to limit the breadth of the invention to the embodimentsillustrated and described. Rather, the scope of the invention is to beinterpreted by the appended claims and their equivalents. Eachpublication, patent, patent application, and reference cited herein ishereby incorporated herein by reference.

What is claimed is:
 1. A method of removing media and cells from a cellculture device, the method comprising: using an apparatus to reduce avolume of a liquid media in the cell culture device containing cells andliquid media, said apparatus comprising; a gas delivery component, afirst fluid detection component that is capable of determining when aliquid media moving within a media removal conduit through a mediaremoval port to the cell culture device is replaced by a gas, said firstfluid detection component capable of sending a signal to a first fluidflow control component that is capable of terminating the fluid flowthrough the media removal conduit, a second fluid detection componentthat is capable of determining when a liquid media moving within a cellremoval conduit that is connected to the cell culture device is replacedby a gas, said second fluid detection component capable of sending asignal to a second fluid flow control component that is capable ofterminating the fluid flow through the cell removal conduit; said cellculture device comprising a gas permeable bottom upon which cellsreside, a media removal conduit and a media removal port, a cell removalconduit and a cell removal port, and a filter; increasing theconcentration of cells per milliliter of media within said cell culturedevice by connecting said gas delivery component to said filter,connecting said first fluid detection component to said media removalconduit, connecting said first fluid flow control component to saidmedia removal conduit, initiating gas delivery from said gas deliverycomponent, whereby gas moves into said cell culture device and displacesthe liquid media from said cell culture device into a media collectionvessel connected to said media removal conduit, said first fluiddetection component determines when the liquid media that is movingthrough said media removal conduit is replaced by gas and upon makingthat determination said first fluid detection component sends a signalto said first fluid flow control component which then terminates theflow of fluid through said media removal conduit; removing cells fromsaid gas permeable cell culture device by connecting said second fluiddetection component to said cell removal conduit, connecting said secondfluid flow control component to said cell removal conduit, andinitiating gas delivery from said gas delivery component, whereby gasmoves into said cell culture device and displaces liquid media and cellsfrom said cell culture device into a cell collection vessel connected tosaid cell removal conduit, said second fluid detection componentdetermines when the fluid that is moving through said cell removalconduit is replaced by gas and sends a signal to said second fluid flowcontrol component which then terminates the flow of fluid through saidcell removal conduit.
 2. The method of claim 1 wherein after said secondflow control component has terminated the flow of fluid through saidcell removal conduit, the method further comprising; venting said cellculture device to atmosphere, said first flow control component isopened to allow the flow of fluid through said media removal conduit,liquid media is moved through said media removal conduit into said cellculture device, said first flow control component is closed, and saidcell culture device is no longer vented to atmosphere, and deliveringgas from said gas delivery component into said cell culture device todisplace liquid media and cells from said cell culture device into acell collection vessel connected to said cell removal conduit, saidsecond fluid detection component determining when the liquid media thatis moving through said cell removal conduit is replaced by gas and uponmaking that determination said second fluid detection component sends asignal to said second fluid flow control component and upon receivingsaid signal, said second flow control component terminates the flow offluid through said cell removal conduit.
 3. The method of claim 1wherein the gas permeable material is in contact with a gas permeablematerial support.
 4. The method of claim 1 wherein the gas permeablematerial of the cell culture device is comprised of silicone.
 5. Themethod of claim 4 wherein the gas permeable material has a thicknessbetween 0.001 inches and 0.024 inches.
 6. The method of claim 1 whereinthe growth surface of said cell culture device is in a substantiallyhorizontal state when gas displaces liquid media from the cell culturedevice.
 7. The method of claim 1 wherein prior to reducing the volume ofliquid media the lowest point of the liquid media to the highest pointof the liquid media is from 1.0 cm to 10.0 cm.
 8. The method of claim 1wherein said cells are comprised of T cells.
 9. The method of claim 1wherein said cells are comprised of chimeric antigen receptor (CAR) Tcells.
 10. The method of claim 1 wherein prior to the removing cells,the method further comprises adding fresh liquid media to said cellculture device through said media removal conduit and said media removalport.
 11. The method of claim 1 wherein said gas delivery component is adiaphragm pump.
 12. The method of claim 1 wherein the media removalconduit within the cell culture device shares a wall with the cellculture device.
 13. The method of claim 1 wherein the distance betweenthe media removal opening and the growth surface is reduced after thefirst flow control component terminates the flow of fluid through saidmedia removal conduit.
 14. The method of claim 13 wherein the cellculture device includes a cell collection pocket in the growth surfacewhich the media removal conduit resides during the step of collectingcells.
 15. The method of claim 1 wherein prior to the removal of cells,agitating the cell culture device to dislodge cells from the growthsurface.
 16. The method of claim 1 wherein during the step of collectingcells the cell culture device is oriented into a position that placesthe cell removal conduit at the low point of the cell culture device.17. The method of claim 1 wherein a portion of the cell removal conduitis positioned within a pocket within the gas permeable bottom of saidcell culture device.
 18. The method of claim 1 wherein when second fluidflow control component terminates the flow of liquid media through saidcell removal conduit, said gas delivery component terminates gasdelivery.
 19. The method of claim 1 wherein said gas delivery componentincludes a pressure relief valve.